Method for diagnosing a molecular phenotype of a patient suffering from an illness accompanied by chronic inflammation

ABSTRACT

Chronic inflammation is an increasing medical problem area of high socioeconomic significance. The invention relates to a method and a kit for diagnosing a molecular phenotype of a patient suffering from an illness accompanied by chronic inflammation, and to a medicament for treating such a patient. To that end, the gene expression of GATA-3 and/or Tbet in a biological isolate of the patient is measured and used for association with a molecular phenotype of the illness.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC OR ASA TEXT FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

Incorporated by reference in its entirety is a sequence listing incomputer-readable form submitted concurrently herewith and identified asfollows: ASCII (text) file named “49433_SeqListing.txt,” 59,470 bytes,created on Mar. 10, 2015.

BACKGROUND OF THE INVENTION

The invention relates to a method and a kit for diagnosing a molecularphenotype of a patient suffering from an illness accompanied by chronicinflammation as well as a medicament for treating such a patient.

Chronic inflammations constitute an increasing medical problem area ofhigh socioeconomic significance. This includes in particular thefollowing groups of illnesses: autoimmune diseases and diseases from thearea of rheumatic diseases (manifestations among others on the skin,lungs, kidneys, vascular system, nervous system, connective tissue,locomotor system, endocrine system), immediate-type allergic reactionsand asthma, chronic obstructive lung diseases (COPD), arteriosclerosis,psoriasis and contact eczema and chronic rejection reactions after organand bone marrow transplants. Many of these diseases are showing a risingprevalence in the last decades not only in industrial nations, butsometimes around the world. For example, in Europe, North America, Japanand Australia more than 20% of the population suffers from allergicdiseases and asthma. Chronic obstructive lung diseases are currently thefifth most frequent cause of death throughout the world and according tocalculations of the WHO they will represent the third most frequentcause of death in the year 2020. Arteriosclerosis with the secondarydiseases of cardiac infarction, stroke and peripheral arterial diseaseleads the world in morbidity and mortality statistics. Together withneurodermatitis, psoriasis and contact eczema are in general the mostfrequent chronic inflammatory diseases of the skin.

Due to the interactions between environmental factors and a geneticdisposition, which are to date only poorly understood, there aresustained dysregulations of the immune system. In this connection thefollowing common principles can be established for these differentdiseases:

(A) An excessive immune response to what are ordinarily harmlessantigens for human beings. These antigens can be components of theenvironment (e.g. allergens such as pollen, animal hairs, food, mites,chemical substances such as preservatives, dyestuffs, detergents). Inthese cases patients develop an allergic reaction. In the case of e.g.active and passive smokers, chronic pulmonary lung diseases (COPD)develop. On the other hand, the immune system can also react againstcomponents of its own organism, recognize them as foreign and initiatean inappropriate inflammatory response. In these cases an autoimmuneillness develops. In any case, harmless, non-toxic antigens areerroneously as foreign or dangerous and an inappropriate inflammatoryresponse is set in motion.

(B) The diseases run in phases, including initiation, progression of theinflammatory response and the associated destruction and reconstructionwith loss of organ functionality (so-called remodeling).

(C) The diseases show patient-specific sub-phenotypic manifestations.

(D) Components of the innate and acquired immunity have a sustainedinvolvement in the initiation, maintenance and destructive andremodeling processes. Under the influence of the innate immunity(important components: antigen-presenting cells with their diversepopulations and the complement system) there is an activation anddifferentiation of the cells of the adaptive immune system (importantcomponents: T and B lymphocytes)>The T cells take over central functionsin the further course by differentiating in highly specializedeffectors.

In this connection they activate and acquire certain effectormechanisms, including, in particular the following functions: antibodyproduction: control of the functionality of effector cells of the immunesystem (e.g. such as neutrophilic, basophilic, eosinophilicgranulocytes), feedback to functions of the innate immune system,influencing of the functionality of non-hematopoietic cells such as e.g.epithelial, endothelial, connective tissue, bones and cartilage andabove all neuronal cells. Here there is a special interaction betweenimmune and nervous systems, from which the concept ofneuro-immunological interaction in the case of chronic inflammationsdeveloped.

Since the T cells, which have already been mentioned, take over centralfunctions in the course of the disease, an understanding of theirspecialization is critical. A complex signal transduction cascade isinvolved in the differentiation of naïve CD4⁺ cells to Th1 or Th2 cells.

The stimulation via the T cell receptor through the correspondingpeptide MHC complex induces clonal expansion and programmeddifferentiation of CD4⁺ T lymphocytes to T helpers (Th) 1 or Th2 cells.The differentiation of these two sub-types occurs on the basis of theircytokine profiles. Th1 cells produce interferon-^(γ) (INF^(γ)),interleukin 2 (IL-2) and tumor-necrosis-factor-α, while Th2 cellssecrete IL-4, IL-5, IL-9 and IL-13. Bacterial and viral infectionsinduce an immune response which is dominated by Th1 cells. On the otherhand Th2 cells regulate igE production against parasites. In the processthere is a balance between Th1 and Th2 cells. The destruction of thisbalance causes diseases, so an excessive Th1 cell response is associatedwith autoimmunity diseases, while allergic diseases are at the basis ofa reinforced Th2 cell response.

It is known that Th1 cytokines are involved in the pathogenesis ofautoimmune diseases such as e.g. autoimmune uveitis, experimentalallergic encephalomyelitis, type 1 diabetes mellitus or Crohn's disease,while Th12 cytokines (IL-4, IL-5, IL-13 or IL-9) are involved in thedevelopment of chronic inflammatory respiratory ailments, such as e.g.airway eosinophilia, asthma, mucus hypersecretion and airwayhyperresponsiveness. These diseases are brought about bypathophysiological changes during the production of characteristiccytokines by antigen-specific Th cells. Th2 cell sub-populations in thelungs and the airways cause the characteristic symptoms of bronchialasthma in the animal model

Among other things, two transcription factors are involved in thedevelopment of autoimmune diseases and chronic inflammatory reactions:the Th1 cell-specific transcription factor Tbet and the Th2cell-specific transcription factor GATA-3.

The Th1 cell-specific transcription factor Tbet is primarily responsiblefor the differentiation of naïve CD4⁺ T cells to Th1 cells. Itsexpression is controlled via the signal transduction pathways of the Tcell receptor (TZR) and via INF^(γ) receptor/STAT1. Tbet transactivatesthe endogenous INF^(γ) gene and induces INF^(γ) production. The in vivofunction of Tbet is confirmed in knock-out mice (Tbet−/−). The quantityof Th2 cytokines is increased in mice that are deficient in Tbet.

The function of Tbet in mucosal T cells is known in the development ofinflammatory bowel diseases. The transcription factor Tbet specificallyinduces the development of Th1 cells and controls the INF^(γ) productionin these cells. Through the inhibition of Tbet the balance between Th1and Th2 cells is shifted in favor of the Th2 cells.

Many inflammatory diseases on the other hand, such as allergic asthmafor example, are associated with an activation of Th2 cells. Th2 cellshave an essential function in the development of allergic diseases, inparticular various asthma ailments. The differentiation of Th0 cells toTh2 cells necessary for this is dependent on the transcription factorGATA-3. GATA-3 is a member of the GATA family of transcription factors.

The Th2 cell-specific transcription factor GATA-3 is primarilyresponsible for the differentiation of naïve CD4⁺ T cells to Th2 cells.In the process, the Th2 cell differentiation is primarily controlled bytwo signal transmission pathways, the T cell receptor (TZR) and the IL-4receptor pathway: Signals forwarded from TZR activate the Th2cell-specific transcription factors cMaf and GATA-3 as well as also thetranscription factors NFAT and AP-1. The activation of the IL-4 receptorresults in the binding of STAT6 on the cytoplasmic domain of the IL-4receptor, where it is phosphorylated by Jak1 and Jak3 kinases. Thephosphorylation for its part results in the dimerization andtranslocation of STAT6 to the nucleus, where STAT6 activates thetranscription of GATA-3 and other genes. GATA-3 is a zinc fingertranscription factor which is expressed exclusively in mature Th2 cells,not in Th1 cells.

Th2 cells produce cytokines such as for example IL-4, IL-5, IL-6, IL-13and GM-CSF. The polarization to Th2 inhibits a Th1 differentiationthrough suppression of Tbet and vice versa. However, the expression ofGATA-3 is not restricted to T cells. An expression of GATA-3 was alsoable to be confirmed in eosinophilic and basophilic granulocytes, mastcells and epithelial cells. GATA-3 plays a central role in theimmunopathogenesis of chronic inflammatory diseases, in particular ofallergic asthma.

Established preparations for the treatment of chronic inflammatorydiseases are among others Corticosteroids, anti-leukotrienes,immunosuppressives and Anti-IgE monoclonal antibodies. Asthma patientshowever respond with varying degrees of success to these therapeuticagents. For a long time the question of what these differences ineffectiveness were to be attributed to has remained unresolved. As aconsequence, the appropriate therapy had to be fine-tuned on the patientmore or less in accordance with the principle of “trial and error”.

However, only recently was it determined that patients suffering fromasthma, for example, could be further divided into subgroups (Woodruffet al., 2009, T-helper Type 2-driven inflammation Defines MajorSubphenotypes of Asthma, Am J RespiCrit Care Med, Vol 180, 388-395).Thus, it was shown that there are at least two sub-groups of asthmapatients, which were designated as “Th2 high” and “Th2 low”. Thesubgroup “Th2 high” in the process has an increased expression of thePOSTN gene, which codes for the protein periostin, as well as the genesfor IL-3 and IL-5. The group “Th2 low” of tested asthma patients shows alow POSTN gene expression, comparable to a control group of healthypersons. These differing molecular phenotypes could be one cause for thedifferent effectiveness of common therapeutic agents. Thus for thesubgroup “Th2 high” an improved treatment response to treatment withcorticosteroids was determined.

It was also found that two groups of asthma patients, namely “Th2 high”and “Th2 low”, respond with varying degrees of success differently to atherapy with a humanized monoclonal antibody to IL-13 (Corren et al.,2011, Lebrikizumab Treatment in Adults with Asthma, The New EnglandJournal of Medicine, 10.1056/NEJMoa 1106469). In the process, anempirical classification of asthma patients in the group “Th2 high”occurred first, when the values for serum-IgE were higher than 100 IU/mland the number of eosinophilic granulocytes was at 0.14×10⁹ cells perliter or greater. With corresponding values below these patients wereplaced in the group “Th2 low”. Alternatively, there was a classificationby the serum periostin level, which serves as a surrogate marker for Th2cytokine IL-13, which is difficult to establish in blood or airwaysamples. In the process, the fact that IL-13 among others induces invitro the expression of the periostin coding gene POSTN in epithelialcells (Woodruff et al., 2007, Proc Natl Acad Sci USA, 104(40): 15858-63.Genome-wide profiling identifies epithelial cell genes associated withasthma and with treatment response to corticosteroids). In accordancewith Corren et al., 2011, patients with a serum periostin level abovethe average were placed in a “periostin high” group. For the mentionedsub-groups “Th2 high” and “Periostin high” a better treatment responseto treatment with Anti-IL-13 antibodies by tendency was described.

According to WO 2009/124090 A1, a certain classification of asthmapatients is likewise proposed, wherein the gene expression of aplurality of candidate genes, such as for example POSTN, CLCA1 andSERPINB2 is employed. Since it is known that this gene is highlyregulated by the Th2 cytokine IL4 or IL-13, the cluster is also referredto as “IL-4/IL-13 signature”. Along with the measurement of the serumperiostin level as well as the corresponding mRNA quantity, in theprocess a determination of the values for serum IgE and the number ofeosinophilic granulocytes were also described.

One disadvantage of patient stratification on the basis of this geneexpression, above all of POSTN, is the fact that along with an“IL-4/IL-13 signature”, the cytokine IL-5 also plays a significant rolein the genesis of asthma. In addition, the role of the protein periostinin the immune cascade and thus the pathogenesis is unknown.

Thus the problem arises of finding a biomarker that is suitable forreliable and simultaneously clinically practicable molecular phenotypingof a human patient who is suffering from a disease that is accompaniedby chronic inflammations in the groups “Th2 high” or “Th2 low” or “Th1high” or “Th1 low”. In addition, the patient classified in this mannershould be able to be treated with a therapeutic agent that is especiallyeffective specifically for this subgroup. The biomarker should makepossible/facilitate an individual prediction about the effectiveness ofa therapeutic agent with respect to a patient, in particular an asthmapatient.

BRIEF SUMMARY OF THE INVENTION

In accordance with the invention, the problem is solved by a method fordiagnosing a molecular phenotype of a human patient suffering from anillness accompanied by chronic inflammation, wherein the molecularphenotype is selected from the group consisting of the subgroups “Th2high”, “Th2 low”, “Th1 high” and “Th1 low” and the gene expression ofGATA-3 and/or Tbet is measured in a biological isolate of the patientand used for the assignment to a molecular phenotype of the illness. Themore detailed classification of a human patient suffering from anillness accompanied by chronic inflammation occurs in the process bymeasurement of the gene expression of the transcription factor GATA-3and/or of the transcription factor Tbet. As explained initially, the Th1cell-specific transcription factor Tbet and the Th2 cell-specifictranscription factor GATA-3 are involved in the development ofautoimmune illnesses and chronic inflammation reactions. Thepolarization to Th2 inhibits a Th1 differentiation through suppressionof Tbet and vice versa. Depending on the expression level of GATA-3and/or Tbet an assignment to a molecular phenotype, that is, to asubgroup of the illness accompanied by chronic inflammation, can takeplace. With the inventive diagnostic method the mentioned molecularphenotyping can be performed without difficulties in a routine clinicalsetting with a high degree of predictability.

The transcription factors GATA-3 and Tbet are the central key moleculesin the development of Th1 or Th2 dependent chronic inflammatorydiseases. Therefore, the direct measurement of the protein or mRNAexpression represents best possible patient stratification approachsince no interconnected mechanisms can possible falsify the results.

In accordance with one preferred embodiment of the method, theexpression level of GATA-3 and/or Tbet is determined via the protein ormRNA quantity. In the process, the protein quantity can bequantitatively determined with the help of an immunoassay. Theimmunoassay is preferably an enzyme-linked immunosorbent assay (ELISA)test, a radioimmunoassay (RIA), an electrochemiluminescence (ECL)immunoassay, a CLIA (chemoluminescence-linked immunosorbent assay), anFLIA (fluorescence-linked immunosorbent assay) or a multiplex-assay.

Along with high sensitivity and specificity, the mentioned assays offerthe advantage of a potential automation and are thus particularly wellsuited for daily clinical practice. Of course, if necessary any othersuitable test for quantitative determination of the protein quantity ofGATA-3 and/or Tbet can be selected within the scope of the presentinvention. Furthermore, the expression level of GATA-3 and/or Tbet canoccur via mass spectrometric methods, chromatographic methods such asgas chromatography, fluid-based methods with solid phase separation,such as HPLC, or microfluidic and nanofluidic methods.

The method for determining the expression of GATA-3 or Tbet with thehelp of an ELISA test can if necessary comprise the following:

-   -   Production of a lysate through cell disruption;    -   Addition of the lysate to a recess of a microwell plate which is        coated with a first GATA-3 or Tbet specific antibody    -   Washing the microwell plate;    -   Addition of a second GATA-3 or Tbet specific antibody to the        recess of the microwell    -   Washing the microwell plate    -   Detection and quantification of the GATA-3 or Tbet protein.

For the purpose of detection, the second specific antibody can forexample be marked with biotin and a separate addition of an enzymecoupled to streptavidin can take place. However, the second specificantibody can also be directly coupled to an enzyme. If appropriate, athird antibody directed toward the second specific antibody can be usedthat is coupled to an enzyme.

The enzyme is preferably a peroxidase or alkaline phosphatase and isimplemented with a suitable substrate that is suitable for colorimetryor chemiluminescence and the like.

In accordance with a further aspect of the present invention, the mRNAquantity of GATA-3 and/or Tbet can be determined additionally or as analternative to the mentioned determination of the protein quantity.Preferably a PCR, particularly preferably a qPCR or a micro-array chipis suitable for this purpose. A person skilled in the art is aware ofhow to select GATA-3 and Tbet specific probes or primers for thementioned detection methods.

In accordance with a preferred design of the method, the biologicalisolate was obtained whole blood, urine, sputum, a bronchial alveolarlavage (BAL), a biopsy, a brush biopsy, liquor, tracheal secretion,seminal fluid, ascitic fluid, saliva, punctate or lymph fluid. A personskilled in the art is familiar with the routine methods for obtainingsuitable biological isolate.

GATA-3 and Tbet are proteins which, as transcription factors, have theireffect in the cell core of T helper cells of the subtype Th1 and T1h2.In order to determine the concentration of these two nuclear proteins ina specified volume of a biological isolate, as in a specified volume ofwhole blood or the like, cells which form GATA-3 and Tbet must first beisolated and subsequently lyzed. A direct confirmation of these proteinsfrom human serum or plasma is hardly possible, since they are notavailable there in detectable concentration. An analysis of GATA-3 andTbet therefore takes place if necessary in four stages:

-   -   Partitioning and isolation of the GATA-3/Tbet expressing cells        from the other cellular components of the whole blood    -   Disruption of the cells and release of the intracellular/nuclear        proteins    -   Measurement of the concentration of GATA-3 and Tbet and    -   Standardization of the found concentrations of GATA-3 and Tbet.

According to an advantageous development, regardless of whether theexpression level of GATA-3 and/or Tbet is determined via the protein ormRNA quantity, the inventive method comprises also one or more of thefollowing steps:

-   -   (i) Isolation of leukocytes, preferably by means of Ficoll        gradient centrifugation;    -   (ii) Enrichment of leukocytes, preferably by means of size        exclusion filtration or    -   (iii) Enrichment of Th1/Th2 cells in particular CD4⁺ T cells        with the help of cell-specific antibodies which are preferably        coupled to magnetic beads.

The mentioned steps (i)-(iii) are performed prior to the cell disruptionand in each case facilitate an increase in the sensitivity as well aspredictability of the diagnostic method, since, in particular in theleukocytes the genes GATA-3 and Tbet are differentially expressed.

According to a further aspect of the present invention an assignment ofthe patient to a molecular phenotype of the subgroup “Th2 high” occurswhen at least one of the following conditions is fulfilled:

-   -   a) The GATA-3 gene expression in the biological isolate is        higher than a defined reference value    -   b) The ratio of GATA-3:Tbet gene expression in the biological        isolate is higher than a defined reference value.

The subgroup “Th2 high” is thus characterized either by a high absoluteGATA-3 gene expression in comparison to a defined reference value. Inthe process,—in the case of the measurement of the protein quantity—acorresponding value of the GATA-3 protein content in the isolate of ahealthy person can be used as a reference value. However, absolutereference values can also be used. In the case of the measurement of themRNA quantity a corresponding value of the GATA-3 mRNA quantity in theisolate of a healthy person can be used as a reference value. However,on the other hand, absolute reference values such as for examplecopies/ml can also be used.

An assignment of the patient to the mentioned molecular phenotype of thesubgroup “Th2 high” takes place according to an advantageous embodimentof the inventive method when as an alternative to or in addition to theincreased GATA-3 gene expression the ratio of GATA-3:Tbet geneexpression in the biological isolate is higher than a defined referencevalue. In the process a corresponding value of the ratio of GATA-3:Tbetgene expression in the isolate of a healthy person is used. Thedetermination of the ratio of GATA-3:Tbet gene expression increases thecertainty of the statement, since in the process along with the GATA-3gene expression the Tbet gene expression is established as an additionalparameter. Since the two transcription factors mutually regulate oneanother in their expression, as initially described, the inclusion ofTbet constitutes an internal control for the measurement of the GATA-3gene expression.

According to one advantageous development, the inventive method forassigning the patient to the molecular phenotype of the subgroup “Th2high” comprises the steps:

-   -   Release of proteins or RNA from cells of a biological isolate of        the patient;    -   Determination of the expression level of the proteins or of the        mRNA for GATA-3 and/or Tbet;    -   Placement of the patient in the subgroup “Th2 high” when at        least one of the foregoing conditions mentioned under a) or b)        apply.

As an alternative, in addition to the mentioned determination of theGATA-3 and/or Tbet gene expression, a determination of furtherparameters for certain placement/classification in the subgroup “Th2high” can take place. Thus, for example the serum IgE level and thenumber of eosinophilic granulocytes can be measured. An assignment tothe subgroup “Th2 high” takes place additionally whenever the serum IgElevel is higher than 100 IU/ml and/or the number of the eosinophilicgranulocytes is 0.14×10⁹ cells per liter or higher. As an alternative,if required, the concentration in nitric oxide in the exhaled air, thusa determination of the FeNO value can be performed.

Another advantageous embodiment of the inventive method relates to anassignment of the patient to a molecular phenotype of the subgroup “Th2low” when at least one of the following conditions is fulfilled:

-   -   a) The GATA-3 gene expression in the biological isolate is lower        than a defined reference value,    -   b) The ratio of GATA-3:Tbet gene expression in the biological        isolate is lower than a defined reference value.

The subgroup “Th2 low” is thus characterized either by a low absoluteGATA-3 gene expression in comparison to a defined reference value. Inthe case of the measurement of the protein quantity, in the process, acorresponding value of the GATA-3 protein content in the isolate of ahealthy person can be used as a reference value. Here it should be notedthat in the case of a patient of the subgroup “Th2 low” with an illnessaccompanied by chronic inflammations the absolute GATA-3 gene expressionwill regularly be higher than in an isolate of a healthy patient.However, the absolute GATA-3 gene expression can also be lower than inthe case of a healthy person. In any case, the GATA-3 gene expression isalso not as high as described for the subgroup “Th2 high”. Consequently,an assignment of the patient to the subgroup “Th2 low” occurs when saidpatient's GATA-3 protein content in comparison to an isolate of ahealthy person, if at all, is only moderately increased. However, fixedreference values can also be used. In the case of the measurement of themRNA quantity, a corresponding value of the GATA-3 mRNA quantity in theisolate of a healthy person can be used as a reference value, wherein anassignment of the patient to the subgroup “Th2 low” takes place whensaid patient's GATA-3 mRNA quantity is lower than in a correspondingsample of a healthy person or in any event is not significantlyincreased. However, on the other hand fixed reference values can beused.

An assignment of the patient to the mentioned molecular phenotype of thesubgroup “Th2 low” takes place according to one advantageous embodimentof the inventive method, when as an alternative to or in addition to thedecreased lower GATA-3 gene expression, the ratio of GATA-3:Tbet geneexpression in the biological isolate is lower than a defined referencevalue. In the process a corresponding value of the ratio of GATA-3:Tbetgene expression in the isolate of a healthy person can be used as areference value. The determination of the ratio of GATA-3:Tbet geneexpression also increases the certainty of the statement in this case,since in the process along with GATA-3 gene expression as an additionalparameter the Tbet gene expression is determined. Since the twotranscription factors mutually regulate one another in their expression,as initially described, the inclusion of Tbet constitutes an internalcontrol for the measurement of the GATA-3 gene expression.

According to one advantageous further development the inventive methodfor assigning the patient to the molecular phenotype of the subgroup“Th2 low” comprises the steps:

-   -   Release of proteins or RNA from cells of a biological isolate of        the patient;    -   Determination of the expression level of the proteins or of the        mRNA for GATA-3 and/or Tbet;    -   Placement of the patient in the subgroup “Th2 low” when at least        one of the foregoing conditions mentioned under a) or b) apply.

As an alternative, in addition to the mentioned determination of theGATA-3 and/or Tbet gene expression, in turn a determination of furtherparameters for certain placement in the subgroup “Th2 low” can takeplace. Thus, for example the serum IgE level and the number ofeosinophilic granulocytes can be measured. An assignment to the subgroup“Th2 low” takes place additionally whenever the serum IgE level is lowerthan 100 IU/ml and/or the number of the eosinophilic granulocytes isbelow 0.14×10⁹ cells per liter. As an alternative, if required, theconcentration in nitric oxide in the exhaled air, thus a determinationof the FeNO value can be performed.

According to another advantageous embodiment, an assignment of thepatient to a molecular phenotype of the subgroup “Th1 high” occurs whenat least one of the following conditions is fulfilled:

-   -   a) The Tbet gene expression in the biological isolate is higher        than a defined reference value,    -   b) The ratio of Tbet:GATA-3 gene expression in the biological        isolate is higher than a defined reference value.

The subgroup “Th1 high” is thus characterized either by a high absoluteTbet gene expression in comparison to a defined reference value. In thecase of the measurement of the protein quantity, in the process, acorresponding value of the Tbet protein content in the isolate of ahealthy person can be used as a reference value, wherein an assignmentof the patient to the subgroup “Th1 high” takes place when saidpatient's Tbet protein content is increased. However, fixed referencevalues can also be used. In the case of the measurement of the mRNAquantity, a corresponding value of the Tbet mRNA quantity in the isolateof a healthy person can be used as a reference value, wherein anassignment of the patient to the subgroup “Th1 high” takes place whensaid patient's Tbet mRNA quantity is increased. However, on the otherhand, fixed reference values can also be used.

An assignment of the patient to the mentioned molecular phenotype of thesubgroup “Th1 high” takes place according to one advantageous embodimentof the inventive method, when as an alternative to or in addition to thedecreased Tbet gene expression, the ratio of Tbet:GATA-3 gene expressionin the biological isolate is higher than a defined reference value. Inthe process a corresponding value of the ratio of Tbet:GATA-3 geneexpression in the isolate of a healthy person can be used as a referencevalue. The determination of the ratio of Tbet:GATA-3 gene expressionalso increases the certainty of the statement in this case, since in theprocess along with the Tbet gene expression as an additional parameterthe GATA-3 gene expression is determined and the inclusion of GATA-3constitutes an internal control for the measurement of the Tbet geneexpression.

According to one advantageous development, the inventive method forassigning the patient to the molecular phenotype of the subgroup “Th1high” comprises the steps:

-   -   Release of proteins or RNA from cells of a biological isolate of        the patient;    -   Determination of the expression level of the proteins or of the        mRNA for Tbet and/or GATA-3;    -   Placement of the patient in the subgroup “Th1 high” when at        least one of the foregoing conditions mentioned under a) or b)        apply.

A further aspect of the present invention relates to a method thatfacilitates an assignment of the patient to a molecular phenotype of asubgroup “Th1 low” when at least one of the following conditions isfulfilled:

-   -   a) The Tbet gene expression in the biological isolate is lower        than a defined reference value,    -   b) The ratio of Tbet:GATA-3 gene expression in the biological        isolate is lower than a defined reference value.

The subgroup “Th1 low” is thus characterized either by a low absoluteTbet gene expression in comparison to a defined reference value. In thecase of the measurement of the protein quantity, in the process, acorresponding value of the Tbet protein content in the isolate of ahealthy person can be used as a reference value. Here it should be notedthat in the case of a patient of the subgroup “Th1 low” with an illnessaccompanied by chronic inflammations the absolute Tbet gene expressioncan however be higher than in an isolate of a healthy person. However,the absolute Tbet gene expression can also be lower than in the case ofa healthy person. In any event, the Tbet gene expression is not as highas described for the subgroup “Th1 high”. Hence, an assignment of thepatient to the subgroup “Th1 low” takes place when said patient's Tbetprotein content in comparison to an isolate of a healthy person, if atall, is increased, however not significantly. However, fixed referencevalues can also be used. In the case of the measurement of the mRNAquantity, a corresponding value of the Tbet mRNA quantity in the isolateof a healthy person can be used as a reference value, wherein anassignment of the patient to the subgroup “Th1 low” takes place whensaid patient's Tbet mRNA quantity is lower than in the correspondingsample of a healthy patient or in any event is not significantlyincreased. However, on the other hand, fixed reference values can alsobe used.

An assignment of the patient to the mentioned molecular phenotype of thesubgroup “Th1 low” takes place according to one advantageous embodimentof the inventive method, when as an alternative to or in addition to thedecreased Tbet gene expression, the ratio of Tbet:GATA-3 gene expressionin the biological isolate is lower than a defined reference value. Inthe process a corresponding value of the ratio of Tbet:GATA-3 geneexpression in the isolate of a healthy person can be used as a referencevalue. The determination of the ratio of Tbet:GATA-3 gene expressionalso increases the certainty of the statement in this case, since in theprocess along with the Tbet gene expression as an additional parameterthe GATA-3 gene expression is determined and the inclusion of GATA-3constitutes in a certain sense an internal control for the measurementof the Tbet gene expression.

According to one advantageous development, the inventive method forassigning the patient to the molecular phenotype of the subgroup “Th1low” comprises the steps:

-   -   Release of proteins or RNA from cells of a biological isolate of        the patient;    -   Determination of the expression level of the proteins or of the        mRNA for Tbet and/or GATA-3;    -   Placement of the patient in the subgroup “Th1 low” when at least        one of the foregoing conditions mentioned under a) or b) apply.

As an alternative, in addition to the mentioned determination of theGATA-3 and/or Tbet gene expression, in turn a determination of furtherparameters for certain placement in the subgroup “Th1 high” and “Th1low” can take place. Thus, for example the number of eosinophilicgranulocytes or the serum IgE level can be measured. An assignment tothe subgroup “Th1 high” takes place when the serum IgE level is lowerthan 100 IU/ml and/or the number of the eosinophilic granulocytes isbelow 0.14×10⁹ cells per liter. Otherwise, an assignment to the subgroup“Th1 low” takes place. As an alternative, if required, the concentrationin nitric oxide in the exhaled air, thus a determination of the FeNOvalue can be performed.

In order to consider differences in sample preparation, astandardization of the concentrations of GATA-3 and Tbet can beperformed. Differences in the sample preparation can for example comeabout through differing cell numbers that are lyzed, through differinglysis efficiencies of the individual samples or through differingcontent in the various cell types within the cell preparations. Inaccordance with the invention, possibilities for standardization includethe following: Standardization to the total protein content of the celllysate, standardization to the cell number that has been lyzed orstandardization to the concentration of specific marker proteins thatare specifically found in specified cell types.

The patients with the diagnosed molecular phenotype of the subgroup “Th2high” can under circumstances simultaneously be placed in the subgroup“Th1 low”. Also, patients with the diagnosed molecular phenotype of thesubgroup “Th1 high” can under circumstances simultaneously be placed inthe subgroup “Th2 low”. This is to be attributed to the fact representedabove that the polarization to Th2 inhibits a Th1 differentiationthrough suppression of Tbet and vice versa.

Within the scope of the present invention, illnesses are diagnosed ortreated that are accompanied by chronic inflammations, such asautoimmune diseases and diseases from the area of rheumatic diseases(manifestations among others on the skin, lungs, kidneys, vascularsystem, nervous system, connective tissue, locomotor system, endocrinesystem), immediate-type allergic reactions and asthma, chronicobstructive lung diseases (COPD), arteriosclerosis, psoriasis andcontact eczema as well as chronic rejection reactions after organ andbone marrow transplants. Also tumor diseases can be diagnosed andtreated in accordance with the invention, provided GATA-3 or Tbet areinvolved in the development and/or deregulated as after-effects.

Within the scope of the present invention, the chronic inflammatorydisease is either Th2-induced, such as for example allergic bronchialasthma, rhinoconjunctivitis, allergic sinusitis, atopical dermatitis,food allergies, pemphigus, ulcerative colitis, parasitic illnesses, orTh1-induced, such as for example psoriasis, allergic contact eczema,Crohn's disease, COPD, rheumatoid arthritis, autoimmune diseases, type 1diabetes mellitus or MS.

The aforementioned problem is additionally solved in accordance with theinvention through a medicament for the treatment of illnesses of a humanpatient with a molecular phenotype that are accompanied by chronicinflammations, wherein the molecular phenotype has been determined inaccordance with one or more embodiments of the mentioned inventivediagnostic method. The identified molecular phenotype comprises in theprocess in particular the groups “Th1 low, “Th1 high”, “Th2 low” or “Th2high”.

According to one preferred embodiment the mentioned medicament containsa specific ribonucleic acid or deoxyribonucleic acid specific for GATA-3or Tbet, in particular a DNAzyme specific for GATA-3 or Tbet.

The “10-23” model represents a general DNAzyme model (Sontoro et al.,1997). DNAzymes of the 10-23 model—also referred to as “10-23 DNAzymes”have a catalytic domain of 15 nucleotides, which are flanked by twosubstrate binding domains. The catalytic domain in the processpreferably has the sequence ggctagctacaacga (SEQ ID No. 154). The lengthof the substrate binding domains is variable: they are either of equallength or variable in length. In one preferred design, the length of thesubstrate binding domains ranges between 6 and 14 nucleotides, veryespecially preferably in each case at least nine nucleotides. SuchDNAzymes comprise the general sequence nnnnnnnnnggctagctacaacgannnnnnnnn(SEQ ID NO 155). Especially preferable in the process are substratebinding domains that bind the mRNA, coding for the proteins GATA-3 andTbet.

The specified catalytic central domain ggctagctacaacga is only onepreferred embodiment. A person skilled in the art is aware of the factthat “10-23 DNAzymes” can be obtained with comparable biologicalactivity with a modified catalytic domain.

In one especially preferred embodiment, the substrate binding domainsare completely complementary to the region that flanks the cleavingsite. However, in order to bind the target RNA and cleave it, theDNAzyme does not necessarily have to be completely complementary.DNAzymes of the 10-23 type cleave the target mRNA on purine-pyrimidinesequences. Within the scope of the present invention the DNAzymespreferably comprise the in vivo active DNAzymes against GATA-3 and Tbetin accordance with WO 2005/033314 A2, whose content is incorporated asdisclosure content of the present invention.

A medicament for specific inhibition of the GATA-3 expression in vivocontains in particular at least one DNAzyme selected from the groupconsisting of DNAzymes with a sequence in accordance with one of thesequences SEQ ID NO 1 through SEQ ID NO 70. Such a DNAzyme bindspreferably on an mRNA which codes for a human GATA-3 gene with a genesequence selected from the sequences SEQ ID NO 151 (human GATA-3 fromdatabase no.: XM_043124), SEQ ID NO 152 (human GATA-3 from Database No.:X58072) and SEQ ID NO 153 (human GATA-3, sequenced from plasmid pCR2.1).

A medicament for specific inhibition of the GATA-3 expression in vivopreferably contains the DNAzyme hgd40 with the sequence5′-GTGGATGGAggctagctacaacgaGTCTTGGAG (SEQ ID NO 40).

A medicament for specific inhibition of the Tbet expression in vivocontains in particular at least one DNAzyme selected from the groupconsisting of DNAzymes with a sequence according to one of the sequencesSEQ ID NO 71 through SEQ ID NO 148. Such a DNAzyme preferably binds onan mRNA which codes for a human Tbet gene with a gene sequence selectedfrom the sequences SEQ ID NO 149 (human Tbet from the Database No.:NM_013351) and SEQ ID NO 150 (human Tbet sequenced from pBluescript-SK).

A medicament for specific inhibition of the Tbet expression in vivocontains preferably the DNAzyme td69 with the sequence5′-GGCAATGAAggctagctaccaacgaTGGGTTTCT (SEQ ID NO 139) or td70 with thesequence 5′-TCACGGCAAggctagctacaacgaGAACTGGGT (SEQ ID No 140).

As an alternative to the DNAzymes the medicament for specific inhibitionof the GATA-3 or Tbet expression can contain a suitable siRNA.

The medicament preferably has a formulation with which the mentionedspecific ribonucleic acid or deoxyribonucleic acid molecules can beadministered to the patient in the form of a pharmaceutically acceptablecomposition either orally, rectally, parenterally, intravenously,intramuscularly or subcutaneously, intracisternally, intravaginally,intraperitoneally, intrathecally, intravascularly, locally (powder,ointment or drops) or in the form of a spray. For the localadministration of the medicament of this invention include ointments,powders, sprays or inhalants. The active component is mixed understerile conditions with a physiologically acceptable excipient andpossible preservatives, buffers or propellants, depending onrequirements.

The medicament can be used for therapy for all diseases that areaccompanied by chronic inflammations.

According to an especially preferred design of the present invention,the treatment of the patients takes place with a molecular phenotype ofthe subgroup “Th2 high” with a GATA-3 specific DNAzyme. The therapy of apatient with a molecular phenotype of the subgroup “Th1 high” takesplace with a Tbet specific DNAzyme. In addition, the treatment of apatient with the molecular phenotype of the subgroup “Th2 low” can takeplace with a Tbet specific DNAzyme and the treatment of a patient withthe molecular phenotype of the subgroup “Th1 low” can occur with aGATA-3 DNAzyme.

An inventive medicament with a GATA-3 specific DNAzyme is thus providedpreferably for the treatment of a patient with the molecular phenotypeof the subgroup “Th2 high” and a medicament with a Tbet specific DNAzymeis preferably provided for the treatment of a patient with the molecularphenotype of the subgroup “Th1 high”.

In accordance with the invention, one special advantage of themedicament according to an embodiment mentioned above for the specifictherapy of an inventively diagnosed subgroup of patients—namely “Th2high”, “Th2 low”, “Th1 high” or “Th1 low” lies in the fact that with thehelp of the specific medicament, in particular of a DNAzyme and/or ansiRNA, a functional inactivation of the coding ribonucleic acidmolecules of transcription factors takes place, whose differentialexpression was determined beforehand and which is involved in thedevelopment of the chronic inflammatory reactions and autoimmunediseases. This strategy differs distinctly from conventional approachesand also differs from the approach according to Corren et al., 2011,since there, one the one hand for example the quantity of periostin(surrogate marker) is measured, but then a therapy aimed at anothertarget, such as for example IL-13 with the help of an anti-IL13 antibodywas proposed. The inventive medicament on the other hand has a highspecificity. It causes a cell-specific intervention and is specific forcompartments and organs.

Dosage forms of the inventive medicament comprise pharmaceuticallyacceptable compositions which contain modifications and “prodrugs”,provided they do not trigger excessive toxicity, irritations or allergicreactions in patients according to reliable medical assessment. The term“prodrug” relates to compounds that are transformed for improvement ofthe absorption, such as for example through hydrolysis in the blood.

The inventive medicament can also be used in the form of a multipleemulsion for application of the mentioned specific nucleic acidmolecules. A suitable multiple emulsion to this end comprises anexterior water phase W1, an oil phase O dispersed in the exterior waterphase W1 and an interior water phase W2 dispersed in the oil phase O,wherein in the interior water phase W2 at least one electrolyte selectedfrom the group of alkali metal halides and earth alkali metal halidesand sulfates and at least one specific ribonucleic acid ordeoxyribonucleic acid molecule, preferably a DNAzyme specific for GATA-3or Tbet is provided, wherein the exterior water phase W1 contains ahydrophilic emulsifier which is a polymer of ethylene oxide andpropylene oxide, and the oil phase O is formed by triacylglycerides andhas a lipophilic emulsifier from the group of dimethicones. With thehelp of such multiple emulsion in particular nucleic acid molecules canbe especially effectively protected from undesirable decomposition.

The type of dosage will be determined by the attending physician inaccordance with the clinical factors. A person skilled in the art isaware of the fact that the type of dosage is dependent on differentfactors such as e.g. body size, weight, body surface, age, sex or thegeneral health of the patient, but also depends on the agent to beadministered, the duration and type of administration and on othermedicaments that may be administered in parallel. In the process,according to an especially advantageous embodiment, the quantity of theactive ingredient of the medicament can be adapted to the measuredexpression level. Thus, in the case of placement in the subgroup “Th2high” and an established very high GATA-3 gene expression an increasedose of the active ingredient, in particular a DNAzyme specific forGATA-3 specific can be administered. Correspondingly, in the case ofplacement in the subgroup “Th1 high” and an established very high Tbetgene expression an increased dose of the active ingredient, inparticular of a DNAzyme specific for Tbet can be administered.

A further aspect of the present invention relates to a kit fordiagnosing a molecular phenotype of a human patient suffering from anillness accompanied by chronic inflammation, wherein the kit contains atleast one specific component for quantitative determination of theprotein or mRNA quantity of GATA-3 and/or Tbet in a biological isolateof the patient.

The inventive kit for diagnosis can be easily offered in the form of aready to use “kit” which comprises antibodies or antigens that areadsorbed on a surface of a carrier and a preparation of human IgGantibodies which e.g. in the case of a human, a preparation ofanti-human IgG antibodies that are marked such that they are proved by acascade of reactions of the type biotin-streptavidin peroxidase oralkaline phosphatase.

As an alternative, the kit for diagnosis also comprises, in addition toa carrier, buffers and reagents, e.g. reagents which are necessary forproof of the reaction such as e.g. streptavidin that is coupled to amarker that gives a color reaction.

As an alternative, the kit additionally comprises a standard sample ofGATA-3 and/or Tbet for calibration of the kit, wherein for proof of theprotein or mRNA of GATA-3 and/or Tbet, a standard sample is used.

In the case of one preferred embodiment, a specific antibody againstGATA-3 and/or Tbet is included for the quantitative determination of theprotein quantity. If necessary, in accordance with a modification,further components for execution of an immunoassay, in particular anELISA, can be included.

The further component for carrying out the ELISA is selected from thegroup consisting of lysis buffers for cell disruption, a microwellplate, protein quantity standards for GATA-3 and/or Tbet, secondaryantibodies and a coupled enzyme for implementation of a substrate fordetection. Preferably, in addition to a first specific antibody againstGATA-3 and/or Tbet the kit comprises a further specific antibody againstGATA-3 or Tbet.

The kit can contain a sequence specific probe and/or primer for theGATA-3 and/or Tbet genes for quantitative determination of the mRNAquantity.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features, details and advantages of the invention arise from thewording of the claims as well as from the following description ofexemplary embodiments with the assistance of the drawings. The figuresshow the following:

FIG. 1 shows the influence of various detergents on the release ofGATA-3 from stimulated Jurkat cells,

FIG. 2a,b show results of a quantification of Tbet and GATA-3 by meansof chromogenic sandwich ELISA,

FIG. 3 shows a standard curve of a GATA-3 ELISA for quantification ofsamples

FIG. 4 shows a standard curve of a Tbet ELISA for quantification ofsamples

FIG. 5 shows a standardized determination of Tbet in lysates of humanperipheral mononuclear cells (PBMC)

FIG. 6 shows a significant improvement of allergic airway inflammationafter four-day treatment with the GATA-3 specific DNAzyme hgd40 (SEQ IDNO 40) compared to untreated mice,

FIG. 7 shows the influence of the GATA-3 specific DNAzyme hgd40 (SEQ IDNO 40) on the number of neutrophils occurring in the chronicinflammation, the number of eosinophils in the BAL and the release ofIL-5 after an eight-week treatment and

FIG. 8 shows the influence of the GATA-3 specific DNAzyme hgd40 (SEQ IDNO 40) on the peribronchial/perivascular inflammation and goblet cellhyperplasia in the lung tissue.

DETAILED DESCRIPTION OF THE INVENTION

Material and Methods:

Cells can be isolated, for example, by means of technologies based onthe binding of specific antibodies. Magnetic beads, which can beobtained from the firms Miltenyi (Macs-System), Dynal (DynaBeads) orBD-Bioscience (iMAG), are used. As an alternative this happens via acell purification by means of fluorescent marked antibodies on cellsorters for example from the firm Cytomation (MOFLO) or BD-Bioscience(FACS-Vantage). The purity of the target cells is preferably at least80%, more strongly preferred at least 95% and most preferred at least99%.

Methods for the isolation of RNA are e.g. described in Sambrook andRussell, Molecular Cloning, A Laboratory Manual, 3^(rd) Edition, ColdSpring Harbor Laboratory (2001), New York and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons (1998), New York. Inaddition it is possible for the average person skilled in the art to usecommercially available kits (Silika-Technologie) e.g. the RNeasy Kitfrom the firm Qiagen, for RNA isolation. In addition it is preferable topurify mRNA directly from the target cells by using commercial kits forexample from the firm Qiagen (Oligotex mRNA Kit), Promega (PolyATractmRNA Isolation System) or Miltenyi (mRNAdirect).

Exemplary Embodiments

Exemplary Embodiment 1

GATA-3 and Tbet are proteins that, as transcription factors, have theireffect in the cell core of T helper cells of the subtype Th1 and Th2. Inorder to determine the concentration of these two nuclear proteins in aspecified volume of a biological isolate in particular in a specifiedvolume of whole blood, cells which form GATA-3 and Tbet must first beisolated and subsequently lyzed. A direct proof of these proteins fromhuman serum or plasma is not possible, since they are not present therein detectable concentration. An analysis of GATA-3 and Tbet thereforetakes place in 4 stages:

-   -   Partitioning and isolation of the GATA-3/Tbet expressing cells        from the other cellular components of the whole blood    -   Disruption of the cells and release of the intracellular/nuclear        proteins    -   Measurement of the concentration of GATA-3 and Tbet and    -   Standardization of the found concentrations of GATA-3 and Tbet.        Partitioning and Isolation of the GATA-3/Tbet Expressing Cells        from the Other Cellular Components of the Whole Blood

This can be performed by different methods of variable complexity, inparticular the following steps for partitioning and isolation within thescope of the present invention:

-   -   An isolation of leukocytes from whole blood by means of Ficcoll        density gradient centrifugation with subsequent affinity        purification of the Th1/Th2 cell types by antibodies against        specific surface markers,    -   If necessary, the affinity purification of the Th1/Th2 cell        types by antibodies against specific surface markers can also be        performed as a 1-stage method without prior enrichment of the        leukocytes,    -   If necessary, the isolation of leukocytes through Ficoll density        gradient centrifugation from whole blood suffices in order to        perform a quantification of the proteins GATA-3 and Tbet,    -   If necessary, in place of the Ficoll density gradient        centrifugation a bead-based affinity purification of the Th1/Th2        cell types through antibodies against specific surface markers        in a deep-well plate in the 96 well format can be employed,    -   If required, in place of the Ficoll density gradient        centrifugation a bead-based affinity purification of the        leukocytes through antibodies against specific surface markers        in a deep-well plate in the 96 well format can be employed,    -   If necessary, a hypoosmolar lysis of the erythrocytes can take        place to obtain a leukocyte preparation or    -   If necessary the protein disruption can occur directly from the        whole blood        Disruption of the Cells and Release of the Intracellular/Nuclear        Proteins

This can be achieved through various methods and principles, inparticular within the scope of the present invention the followingprocedural steps:

-   -   Destruction of cellular membranes through lysis buffers with        different principles of operation:        -   a) Hypotonic buffers which induce a bursting of the cells        -   b) Buffers containing detergents, which destroy the cell            membrane and as a result, release intracellular proteins        -   c) Buffers of high ionic strength or osmotically active            buffers which remove water from the cells and as a result            destroy the cell integrity    -   Physical methods such as heating up, shock freezing or        ultrasound    -   Mechanical methods such as homogenizing or grinding.

Examples of buffers containing detergents could be:

-   -   Buffer systems with a high concentration of ionic (e.g. SDS or        cholate and its derivatives) or non-ionic (e.g. triton or        Tween-20) detergents    -   Mixtures of ionic and non-ionic detergents (e.g. Ripa buffers        with 50 mM Tris.HCl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium        deoxycholate and 0.1% SDS)    -   Commercially available lysis buffers with unknown composition        (e.g. M-PER)        The influence of different detergents on the release of GATA-3        from stimulated Jurkat cells is illustrated in FIG. 1. The lysis        of Jurkat cells (human T cell line) through various lysis        buffers and quantification of GATA-3 by means of ELISA resulted        in an especially high release of GATA-3 protein in the case of        the use of the RIPA buffer (1% RIPA). About 50 ng/ml of GATA-3        were verified.        Measurement of the Concentration of GATA-3 and Tbet

In principle, the concentration of the two transcription factors GATA-3and Tbet can be determined with different methods. Within the scope ofthe present invention, among others there are:

-   -   ELISA (enzyme linked immunosorbent assay)    -   CLIA (chemoluminescence linked immunosorbent assay)    -   FLIA (fluorescence linked immunosorbent assay)    -   Mass spectrometric methods    -   Chromatographic methods (e.g. gas chromatography)    -   Fluid-based methods with solid phase separation, (e.g. HPLC)    -   Microfluidic and nanofluidic methods

FIG. 2a and FIG. 2b show the results of a quantification of Tbet andGATA-3 by means of chromogenic sandwich ELISA. The cells were obtainedfrom whole blood through Ficoll density gradient centrifugation. Thecells (stimulated human mononuclear cells) were lyzed with a Ripabuffer. The lysate was examined with two ELISA prototype methods withrespect to the concentration of GATA-3 and Tbet. The concentration ofthe two proteins was depicted with respect to the total proteinconcentration of the cell lysates (standardization to protein content).

The results in accordance with FIG. 2a show that Th1 cells have a highercontent of Tbet (circa 160 ng/ml analyte/mg protein) than Th2 cells(circa 56 ng/ml analyte/mg protein) and this fact can be clearlyconfirmed from the results of the ELISA test:

Ng/ml Tbet/mg Cells Protein Th2 B11-14 51.01 Th2 B11-15 58.40 Th2 B11-1650.24 Th2 B11-17 68.79 Th2 B11-19 49.63 Th2 B11-20 55.85 Mean 55.65STABW 7.31 VK (%) 13.14 Th1 B11-14 202.24 Th1 B11-19 106.34 Th1 B11-20167.46 Mean 158.68 STABW 48.55 VK (%) 30.59

The Tbet content in the Th1 cells is thus more than two times greater,namely by a factor of about three, than in the Th2 cells.

According to FIG. 2b the content in GATA-3 in the Th2 cells (circa 10ng/ml analyte/mg protein) is higher than in Th1 cells (circa 6 ng/mlanalyte/mg protein) The GATA-3 content is more than 1.5 times higher inthe Th2 cells here—namely by a factor of about 1.7—than in Th1 cells.

In addition, one can see from FIGS. 2a and 2b that in the case ofstandardization to the protein content the quantity ratio of Tbet:GATA-3in Th1 cells differs significantly form the corresponding ratio in Th2cells. Thus the quantity ratio of Tbet:GATA-3 here in Th1 cells is about27, thus more than 20, while the corresponding quantity ratio in Th2cells is circa 6, thus less than 10.

The quantitative determination of GATA-3 and Tbet occurs in each case bymeans of a sandwich ELISA (Enzyme linked immune sorbent assay).

Exemplary Embodiment 2—GATA-3 ELISA:

To this end, the wells of a 96 well microwell plate are coated withspecific antibodies against GATA-3. After addition of the sample or of astandard, GATA-3 binds on the antibodies on the 96 well plate. After awash step to remove the non-bound substances a second, specificbiotinylated antibody against GATA-3 is added. After an additional washstep to remove the non-bound substances peroxidase marked streptavidinis added. After a last wash step to remove the non-bound substancessubstrate is added. The color development is terminated after a definedtime by adding a stop solution. The intensity of the color developmentis quantified by a microwell plate reader. The quantification of thesamples occurs by a comparison to the included standards of knownprotein concentration. FIG. 3 shows a corresponding standard curve of aGATA-3 ELISA.

According to the exemplary embodiment for the performance of the GATA-3ELISA, the steps relate to the following in detail:

-   -   Insert number of required wells into a frame of the 96 well        plate    -   Addition of 50 μl/well assay buffer    -   Addition of 100 μl/well standard/control/sample    -   Incubation for 60 minutes on the shaker    -   Wash all wells 4× with 400 μl/well of wash buffer    -   Addition of 100 μl/well biotinylated anti-GATA-3 antibodies    -   Incubation for 60 minutes on the shaker    -   Wash all wells 4× with 400 μl/well of wash buffer    -   Addition of 100 μl/well peroxidase marked streptavidin    -   Incubation for 30 minutes on the shaker    -   Wash all wells 4× with 400 μl/well of wash buffer    -   Addition of 100 μl/well substrate    -   Incubation for 30 minutes    -   Stop reaction by addition of 100 μl stop solution    -   Measurement of optical density at 450 nm with a microwell plate        reader        Exemplary Embodiment 3—Tbet ELISA:

The verification of the Tbet protein is executed in accordance with thefollowing test principle: The quantitative determination of Tbet occursby means of a sandwich ELISA (Enzyme linked immuno sorbent assay). Tothis end the wells of a 96 well microwell plate are coated with specificantibodies against Tbet. After addition of the sample or of a standard,Tbet binds on the antibodies on the 96 well plate. After a wash step toremove the non-bound substances a second, specific antibody against Tbetis added. After an additional wash step to remove the non-boundsubstances a peroxidase marked antibody against the Tbet specificantibody is added. After a last wash step to remove the non-boundsubstances substrate is added. The color development is terminated aftera defined time by adding a stop solution. The intensity of the colordevelopment is quantified by a microwell plate reader. Thequantification of the samples occurs by a comparison to the includedstandards of known protein concentration. FIG. 4 shows a correspondingstandard curve of a Tbet ELISA.

According to the exemplary embodiment for the performance of the TbetELISA the steps relate to the following in detail:

-   -   Insert number of required wells into a frame of the 96 well        plate    -   Addition of 50 μl/well assay buffer    -   Addition of 100 μl/well standard/control/sample    -   Incubation for 60 minutes on the shaker    -   Wash all wells 4× with 400 μl/well of wash buffer    -   Addition of 100 μl/well anti-Tbet antibodies    -   Incubation for 60 minutes on the shaker    -   Wash all wells 4× with 400 μl/well of wash buffer    -   Addition of 100 μl/well peroxidase marked anti-Tbet specific        antibodies    -   Incubation for 30 minutes on the shaker    -   Wash all wells 4× with 400 μl/well of wash buffer    -   Addition of 100 μl/well substrate    -   Incubation for 30 minutes    -   Stop reaction by addition of 100 μl stop solution    -   Measurement of optical density at 450 nm with a microwell plate        reader        Standardization of the Concentrations of GATA-3 and Tbet

In order to consider differences in the sample preparation, astandardization of the concentrations of GATA-3 and Tbet can beperformed. Differences in the sample preparation can arise e.g. due tothe following:

-   -   Differing cell numbers to be lyzed    -   Differing lysis efficiencies of the individual samples or    -   Differing content in the different cell types within the cell        preparations.

Possibilities for standardization include the following:

-   -   Standardization to the total protein content of the cell lysate        (see under “Measurement of the Concentrations of GATA-3 and        Tbet”)    -   Standardization to the cell number being lyzed (see FIG. 5) or    -   Standardization to the concentration of specific marker proteins        that are specifically found in specified cell types.

FIG. 5 shows a standardized determination of Tbet in lysates of humanperipheral mononuclear cells (PBMC). In the process a lysis of PBMCs ofhealthy subjects and patients suffering from allergic illnesses, such ase.g. allergic bronchial asthma, rhinoconjunctivitis, allergic sinusitis,atopical dermatitis, food allergies takes place. The concentration ofTbet was standardized to the cell number in the lysates. The illness isTh2 dependent and consistently a slight concentration in Tbet (circa 12ng/ml/1 million cells) for allergy sufferers was determined compared tohealthy subjects (circa 27 ng/ml/1 million cells). The Tbetconcentration was thus reduced in the case of allergy sufferers by morethan a factor of 2 compared to healthy subjects. Consequently, anassignment of the patients to the molecular phenotype “Th1 low” iseasily possible here, since the Tbet gene expression in the biologicalisolate is lower than a defined reference value, here the Tbet geneexpression of healthy subjects.

Exemplary Embodiment 4

In modification of Examples 2 and 3, in accordance with Example 4Th1/Th2 cells are enriched by means of magnetic beads coated with cellspecific antibodies for the sample preparation. Subsequently theverification of GATA-3 occurred in accordance with the provisionaccording to Example 2.

Exemplary Embodiment 5

In modification of Examples 2 and 3, in accordance with Example 4leukocytes are enriched by means of size exclusion filtration for thesample preparation. Subsequently the verification of GATA-3 occurred inaccordance with the provision according to Example 2.

Exemplary Embodiment 6

A GATA-3 specific DNAzyme shows therapeutic effects in the mouse modelof the OVA induced allergic airway inflammation of the “Th2 high”phenotype.

In order to provide the best possible illustration of the clinicalphenotype “Th2 high” in the mouse model BALB/c mice were sensitized withthe model allergen ovalbumin (OVA) in the presence of the adjuvantAI(OH)₃ on days 0, 14 and 21 through intraperitoneal injection. On days24-26 the mice inhaled a 1% OVA aerosol in order to cause a Th2dominated allergic inflammatory reaction in the lungs. On days 23-26 theGATA-3 specific DNAzyme hgd40 (SEQ ID NO 40), dissolved in PBS, wasintranasally administered. In the process the Balb/c-mouse strain ischaracterized in that it generates preferentially strong Th2 responses.This is reinforced by the use of AL(OH)₃ as an adjuvant, whichdistinctly supports the formation of Th2 dominated immune responses. Thedescribed mouse model is correspondingly characterized by a massiveinfiltration of eosinophils an Th2 cells in the airways accompanied by ahyperplasia of the mucus forming goblet cells with increased mucusproduction as well as the formation of an airway hyperresponsiveness.Immunologically, along with allergen specific Th2 cells, characterizedby the production of the typical cytokines IL-4, IL-5 and IL-13, alsoOVA specific antibodies of the immune globulin classes IgE and IgG1 (inthe mouse both Th2 dependent) were detectable. All these parameters aretypical clinical features of a “Th2 high” phenotype (Wenzel et al., Am JRespir Crit Care Med. 199 September; 160(3):1001-8; Woodruff et al.,2009). In the process the reaction strength with respect to someparameters in the animal model were even more distinctly pronounced thanin the clinical situation with human patients, e.g. eosinophilicgranulocytes constitute circa 60-70% of all leukocytes in the bronchialalveolar lavage (BAL) in the mouse model, while already 3-5% of thesecells in the sputum of patients indicates a Th2 dominated phenotype.

According to FIG. 6, after four-day treatment with the GATA-3 specificDNYzyme hgd40 (SEQ ID NO 40) compared to untreated mice, a significantimprovement of the allergic airway inflammation was ascertained. Aboveall the number of eosinophils in the BAL was significantly reduced. Inaddition, the BAL concentrations of the characteristic cytokines for thephenotype “Th2 high”, IL-5 and IL-13, were able to be significantlyreduced.

Exemplary Embodiment 7

A GATA-3 specific DNAzyme shows significant therapeutic effects in thechronic mouse model of a Th2 dominated allergic airway inflammation.

In order to provide the best possible illustration of the clinicalphenotype “Th2 high” in the mouse model, BALB/c mice were sensitizedwith the model allergen ovalbumin (OVA) in the presence of the adjuvantAI(OH)₃ on days 0, 14 and 21 through intraperitoneal injection. By meansof twice weekly OVA aerosol provocations over a time period of 14 weeksa chronic inflammation of the airways was caused in the mice. During thelast eight weeks therapy was provided three times a week (until day 121)either with budesonide or the GATA-3 specific DNAzyme hdg40 throughintranasal application.

According to FIGS. 7 and 8 after eight weeks of treatment with GATA-3specific DNAzyme hdg40 (SEQ ID NO 40) the number of eosinophils in theBAL was able to be significantly reduced and in addition a reduction ofthe number of neutrophils occurring in the chronic inflammation was alsoobserved. This was accompanied by a lowered peribronchial/perivascularinflammation and reduced goblet cell hyperplasia. Simultaneously, inre-stimulated lymphocytes of those treated with hgd40 a reduced releaseof IL-5 was observed. In the budesonide group, on the other hand nosignificant improvement of the parameters cold be observed.

The invention is not restricted to one of the previously describedembodiments, but rather can be modified in many respects.

All features and advantages arising from the claims, the description andthe drawings, including design details, spatial arrangements andprocedural steps can be essential to the invention both individually aswell as in a variety of combinations.

We claim:
 1. A method for diagnosing and treating chronic inflammationin a patient suffering from an illness associated with chronicinflammation, by identifying a molecular phenotype of the patientwherein the molecular phenotype is selected from the group consisting ofthe subgroups “Th2 high”, and “Th2 low” and the gene expression of Th2cell-specific transcription factor (GATA-3) and/or the Th1 cell-specifictranscription factor (Tbet) is measured in a biological isolate of thepatient and used for the assignment to a molecular phenotype of theillness wherein the expression level of GATA-3 and/or Tbet is determinedvia the protein or mRNA quantity, and wherein the protein quantity isquantitatively determined by an immunoassay and wherein a therapeuticagent effective for specifically inhibiting GATA-3 or Tbet expressionaccording to the identified molecular phenotype is administered to thepatient.
 2. The method according to claim 1, characterized in that theimmunoassay is selected from the group consisting of an ELISA test, aradioimmunoassay, an electrochemiluminescence immunoassay, a CLIA(chemoluminescence-linked immunosorbent assay), an FLIA(fluorescence-linked immunosorbent assay) and a multiplex-assay.
 3. Themethod according to claim 1, characterized in that an assignment of thepatient to a molecular phenotype of the subgroup “Th2 high” occurs whenat least one of the following conditions is fulfilled: the GATA-3geneexpression in the biological isolate is higher than a defined referencevalue the ratio of GATA-3 : Tbet gene expression in the biologicalisolate is higher than a defined reference value.
 4. The methodaccording to claim 1, characterized in that an assignment of the patientto a molecular phenotype of the subgroup “Th2 low” occurs when at leastone of the following conditions is fulfilled: the GATA-3 gene expressionin the biological isolate is lower than a defined reference value, theratio of GATA-3 : Tbet gene expression in the biological isolate islower than a defined reference value.
 5. The method according to claim1, characterized in that along with the determination of the geneexpression of GATA-3 and/or Tbet a determination of the serum IgE leveland/or the number of eosinophilic granulocytes occurs and/or the Fe_(NO)value is determined.
 6. The method according to claim 1, characterizedin that the illness accompanied by chronic inflammations is Th2-induced,such as for example allergic bronchial asthma, rhinoconjunctivitis,allergic sinusitis, atopical dermatitis, food allergies, pemphigus,ulcerative colitzis, parasitic illnesses, or Th1-induced, such as forexample psoriasis, allergic contact eczema, Crohn's disease, COPD,rheumatoid arthritis, autoimmune diseases, type 1 diabetes mellitus orMS.